Higher NDUFS8 Serum Levels Correlate with Better Insulin Sensitivity in Type 1 Diabetes

Aim: The aim of the study was to evaluate the function of Complex I by measuring NADH dehydrogenase [ubiquinone] iron-sulfur protein 8 serum level and the relationship with insulin resistance in type 1 diabetes. T1DM causes adverse changes in the mitochondria, which can influence the development of chronic complications. The NADH dehydrogenase [ubiquinone] iron-sulfur protein 8 (NDUFS8 protein) is a subunit of NADH dehydrogenase and plays an important role in the mitochondrial function. NDUFS8 serum concentration probably reflects the function of mitochondria. Methods: All of 36 people suffer from T1DM. All participants were treated with functional intensive insulin therapy. NDUFS8 protein serum concentration was measured using the ELISA test. Insulin resistance was evaluated with indirect marker estimated glucose disposal rate (eGDR). The group was divided on the base of median value of eGDR (higher eGDR – less IR). Results: The study group consisted of 12 women and 24 men, aged 39.5 (28.0-46.5) years with the duration of the disease 22 (15-26) years. Medians of eGDR and NDUFS8 protein concentration were 7.6 (5.58-8.99) mg/kg/min and 2.25 (0.72-3.81) ng/ml, respectively. The group with higher insulin sensitivity had higher NDUFS8 protein serum concentration, lower WHR, BMI and they were younger. A negative correlation was observed between NDUFS8 protein serum concentration and WHR (rs=-0.35,p=0.03), whereas a positive correlation was observed between NDUFS8 protein serum concentration and eGDR (rs=0.43,p=0.008). Multivariate linear regression confirmed a significant association between insulin sensitivity and better mitochondrial function (beta=0.54,p=0.003), independent of age, duration of diabetes and smoking. Conclusions: Higher NDUFS8 protein serum concentration is associated with higher insulin sensitivity among people with T1DM and might reflects better mitochondrial function.


INTRODUCTION
Type 1 diabetes mellitus (T1DM) is a disorder characterized by destruction of pancreatic β cells. This process leads to complete insulin deficiency 1,2 . The etiology is still being analyzed and prevention possibilities investigated 3 . The incidence of DM is continually increasing all over the world, in the Polish population as well [4][5][6] . Adults with T1DM experience lower health-related quality of life, are more frequently unemployed and have more sick leave per year in contrast to the general population 7 . These facts are attributed to the development of chronic complications: neurovascular and macrovascular 8 . The development of chronic complications is connected with several risk factors, most prominently insulin resistance (IR) 9 and hyperglycemia 10 .
Insulin resistance is mostly connected to obesity and diabetes mellitus type 2.
However, more and more young people diagnosed with T1DM are overweight or obese at the moment of diagnosis 11,12 . Moreover, IR also develops during T1DM as a result of exogenous insulin treatment and aging. Insulin therapy causes abdominal obesity as well as smoking and physical laziness. Widely used indirect marker of IR in DM1 is estimated glucose disposal rate (eGDR), which was created well with IR measured by hyperinsulinemiceuglycemic clamp, the gold diagnostic standard of IR in DM1 13 .
The main pathomechanisms of hyperglycemia based complications other than enhanced glycolysisthe main pathway of glucose metabolism, are increased flux through the polyol pathway, intracellular production of advanced glycation end products (AGE) precursors, protein kinase C (PKC) isoforms activation and increased hexosamine pathway activity. Furthermore, it is also known that IR is strongly associated with micro and macrovascular complications 9,14,15 . All these mechanisms have a common underlying process: increased reactive oxygen species (ROS, free radicals) production 16 .
Oxidative stress is defined as a disturbance in the balance between the production of ROS and antioxidant defenses which leads to cellular damage 17 . The main organelle responsible for ROS production is the mitochondrion. In the inner membrane of this organelle exist four complexes, creating the mitochondrial respiratory chain. This whole structure is responsible for electron transport. During each one of these four steps, partially reduced oxygen intermediates are being generated. Some of them are very stable and can be stored by the enzyme cytochrome c oxidase until all the electrons are transferred.
In hyperglycemic conditions, in cells there is increased glucose oxidation during tricarboxylic acid (TCA) cycle caused by persistent hyperglycemia and enhanced B-oxidation of free fatty acids (in macrovascular endothelial cells) due to insulin resistance. This leads to a situation where more electron donors, mainly NADH, are released and more protons go through the mitochondrial membrane, thus higher voltage is created 21 . As a result, more ROS are produced and released. By damaging mitochondrial DNA they activate poly(ADPribose) polymerase (PARP), which is a cascade of events inhibits glyceraldehyde-3 phosphate dehydrogenase (GAPDH). Since GADPH is a pivotal enzyme of glycolysis, this inhibition causes not only inhibition of this process but also accumulation of upper level of glycolysis' substrates and products, which in the end leads to the processes mentioned before like for example PKC activation and the polyol pathway 16 . As polyol pathway generates NADH, Complex I has to handle more and more NADH, which provides an argument for the assumption that impaired mitochondrial function means increased Complex I's oxidative activity 22 . Nonetheless, it is proven that the redox balance in DM between NADH and NAD + is highly elevated 23,24 , leading to enhanced ROS production, which shows the insufficiency in mitochondrial NADH dehydrogenase activity.
It was proven that ROS production is due to high proton potential 21 . Some trials have been made in patients with T2DM, in which they discovered the possible role of mitochondrial complex I impairment 25 . Complex I is proposed as a responsible source of oxidative stress also among patients with polycystic ovarian syndrome with IR 26 . We found that NADH dehydrogenase [ubiquinone] iron-sulfur protein 8 (NDUFS8 protein) can be measured in human serum. It is a subunit of NADH dehydrogenase (ubiquinone) also known as Complex I encoded by the NDUFS8 gene 27 . The NDUFS8 gene is located on chromosome 11q13. It spans about 6kb and contains seven exons ranging in size from 51 to 186bp.
Expression of the gene is ubiquitous but predominant in heart and skeletal muscle 28 .
Mutations in this gene have been associated with Leigh syndrome (neurodegenerative disorder) 29 . This mutation cause proximal myopathy, cardiomyopathy, sensorineural deafness, optic atrophy, pigmentary retinopathy, encephalopathy, peripheral neuropathy and exercise intolerance 30 . No studies have been made on patients with T1DM. The aim of the study was to evaluate the function of Complex I by measuring NADH dehydrogenase [ubiquinone] iron-sulfur protein 8 serum level and the relationship with insulin resistance in type 1 diabetes. As a marker of mitochondrial function, it can help to find patients more prone to the development of chronic complications like retinopathy or neuropathy and hence start the prevention strategies accurately earlier.

Study Design
The study design: a cross-sectional, single-center study. The study group consisted of 36 adults with T1DM. Participants were recruited during 1 year (2018-2019). Subjects were informed about the aim of the study and signed a consent form. The study was approved by the local Ethical Committee at the Medical University of Poznan (resolution no. 15/18). We confirm that all methods were performed in accordance with the relevant guidelines and regulations. The inclusion criteria were: age above 18 years old, at least five years history of T1DM confirmed in the past with T1DM antibodies antibodies (to glutamic acid decarboxylase -antiGAD, islet cells -ICA, islet tyrosine phosphatase 2 -IA2), whereas the exclusion criteria were: CRP>5mg/l, unstable hypo/hyperthyroidism (TSH beyond normal range), other endocrinological disorders, contagious diseases, renal or liver diseases, pregnancy, antineoplastic therapy in less than 2 years and any psychological or psychiatric disorder. Everyone was treated with functional intensive insulin therapy at the onset of diabetes. This method aims to mimic physiology, two types of insulin are used: long-acting insulin analogues (so-called basal insulin) and rapid-acting analogues (used before main meals) with insulin pen devices or rapid acting analogues in the insulin therapy with personal insulin pumps. All the participants completed a standardized questionnaire including details of age, sex, chronic diseases, medicines, family history regarding diabetes and pack-years of cigarette smoking, duration of diabetes, blood glucose self-control and medical history.
Participants underwent a complete physical examination with anthropometric measurements (weight, height, waist and hip circumference) and blood pressure check.

Insulin resistance markers
Insulin resistance was evaluated with indirect markers (the estimated glucose disposal rate -eGDR, waist to hip ratio -WHR, body mass index -BMI). The clinical characteristics of the whole study group and according to the presence of insulin resistance are shown in Table 1. The estimated glucose disposal rate (eGDR) was calculated by the following mathematical formula: 24,31-12,22 (WHR) -3,29 (hypertension 0/1) -0,57 (HbA1c) [mg/kg/min] 31 , waist to hip ratio (WHR) was checked using the non-elastic measuring tape with the resolution of 0.5 cm and calculated from the following equation: WHR=waist circumference [cm] / hip circumference [cm]. It was assumed that the higher the eGDR, the higher tissue sensitivity for insulin.

Blood tests
Blood samples were collected in a fasting state using the S-Monovette blood collection system. We evaluated: glycated hemoglobin level (A1c), serum total cholesterol, high-density lipoproteins (HDL) cholesterol, low-density lipoproteins (LDL) cholesterol, triglycerides (TG).

NADH dehydrogenase [ubiquinone] iron-sulfur protein 8 measurement
NADH dehydrogenase [ubiquinone] iron-sulfur protein 8 (NDUFS8 protein), mitochondrial (also known as NADH-ubiquinone oxidoreductase 23 kDa subunit, Complex I-23kD (CI-23kD), or TYKY subunit) serum concentration was measured using the ELISA test (this immunoassay kit allows for the in vitro quantitative determination of human NADH dehydrogenase [ubiquinone] iron-sulfur protein 8, mitochondrial, it's concentrations in serum, plasma, urine, tissue homogenates and cell culture supernates and other biological fluids). The NDUFS8 protein is a subunit of NADH dehydrogenase (ubiquinone) also known as Complex I, which is located in the mitochondrial inner membrane and is the largest of the five complexes of the electron transport chain. The reference range for Elisa kit is 0.312-20 ng/ml, sensitivity 0.1 ng/mL.

Statistical analysis
The statistical analysis was performed using the STATISTICA 13.3 program. The normality of distributions was tested using Kolmogorov-Smirnov's test with Lilliefors correction. Due to lack of normality, non-parametrical tests were performed. All data are expressed as medians and IQR-interquartile range or percentage of subjects. Usually, the value of the cut-off point for eGDR is 7.5 mg/kg/min according to the clamp technique of DeFronzo 31 . In this case, the patients were divided into two groups, below and above eGDR median, because a higher eGDR value shows insulin sensitivity better (less IR). The Mann-Whitney U and Chi 2 tests were used to assess differences between groups. The multivariate linear regression, R Spearman correlation were performed. Differences with a probability value <0.05 were considered statistically significant.
Median of NDUFS8 protein concentration was 2.25 (0.72-3.81) ng/ml (Table 1). People with eGDR above the median (less IR) were proved to have higher NDUFS8 protein serum concentration, lower WHR, BMI and they were younger (Table 1).   The negative correlation was observed between NDUFS8 protein serum concentration and WHR (rs= -0.35, p=0.03) (Figure 1), whereas positive correlation was observed between NDUFS8 protein serum concentration and eGDR (rs=0.43, p=0.008) ( Figure 2).  Multivariate linear regression confirmed a significant association between insulin sensitivity and better mitochondrial function (beta = 0.54, p = 0.003), independent of age, duration of diabetes and smoking (Table 2).

DISCUSSION
The study was designed to assess the mitochondrial function in adults with T1DM and its relationship with insulin resistance (IR). We found that people with T1DM who had better insulin sensitivity (eGDR above the median) had higher NADH dehydrogenase [ubiquinone] iron-sulfur protein 8 (NDUFS8) serum concentration. The relationship was independent of other important factors. There is still lack of information on the exact mechanisms of how Complex I function is indeed impaired: increased or decreased in activity among patients with IR neither what is the best way to check it. The relationship is probably two-sided.
There are also no reference range of NDUFS8 protein serum concentration in healthy people, nor in people with T1DM. NDUFS8 protein concentration was measured in serum as a simple, non-invasive way that reflects the function of mitochondria. As a marker of mitochondrial function, it can help us to find patients more prone to the development of IR and hence start the prevention strategies accurately earlier. complex 1 functionality and an increase in ROS production 37 . We therefore assume that a higher concentration of single subunits determines good performance and better mitochondrial function.
Oxidative stress is the process that is known as the one that causes IR and in general may be involved in T1DM pathogenesis 38 . Oxidative stress induces insulin resistance and arises from chronic low-grade inflammation due to increased amounts of free fatty acids (FFA) and pro-inflammatory cytokines released from adipose tissue 39,40 . Also there is an increased flux of FFA from adipocytes, which then undergo B-oxidation. In this process NADH is produced as well as Aceto-CoA. NADH goes to electron transport chain together with NADH produced by tricarboxylic acid (TCA) cycle started by the delivery of Acetyl-CoA from B-oxidation. It is exactly the same mechanism of ROS production as hiperglycemia causes.
Free radicals damage DNA, proteins and lipids 41 48 . The activity of complex I was performed by an in-gel based blue native polyacrylamide gel electrophoresis (BN-PAGE) analysis which allows to analyze highmolecular-weight protein complexes such as the ones building the mitochondrial inner membrane 49 . As a proof of complex I hyperactivity, it is also reported that metformin, a widely used drug to treat IR among people with diabetes, inhibits Complex I as one of it's main mechanisms to help reducing IR 50 . These studies showed that IR development and mitochondrial damage are an ongoing process.
There are studies that tried to find out which exactly part of the electron transport chain is responsible for the augmented ROS production that drives the described spiral of unwanted events in T1DM patients' cells. T1DM mice model has shown that mitochondria with single nucleotide polymorphism in the mitochondrial gene encoding NADH dehydrogenase subunit 2 (mt-ND2) are characterized by lower reactive oxygen species production and are more resistant to nitric oxide 51 . Another study showed that subjects with three mtDNA RFLPs (restriction fragment length polymorphism ) (morphs), two in the subunit 5 of the NADH dehydrogenase gene and one in the tRNA for threonine, were characterized a higher maximal oxygen uptake (VO2max) in the untrained state than noncarriers. VO2 max is a marker associated with insulin sensitivity. Studies carried out in healthy people suggest that certain mtDNA variants may contribute to increasing in VO2max and its response to training 52 .
We assume that higher NDUFS8 protein concentration confirms good mitochondrial function. In this case the results proved an association between good mitochondrial oxidative function and better insulin sensitivity. Therefore by diminishing IR there is a possibility to influence and improve the mitochondrial function, meaning also delay the